Background and Objectives: Raw electroencephalographic (EEG) signals are rarely used to monitor the depth of volatile induction of general anaesthesia (VIGA) with sevoflurane, even though EEG-based indices may show aberrant values. We aimed to identify whether response (RE) and state entropy (SE) variations reliably reflect the actual depth of general anaesthesia in the presence of different types of epileptiform patterns (EPs) in EEGs during induction of general anaesthesia. Materials and Methods: A randomized, prospective clinical study was performed with 60 patients receiving VIGA using sevoflurane with the increasing concentrations (group VIMA) or the vital capacity (group VCRII) technique or an intravenous single dose of propofol (group PROP). Facial electromyography (fEMG), fraction of inspired sevoflurane (FiAA), fraction of expired sevoflurane (FeAA), minimal alveolar concentration (MAC) of sevoflurane, RE and SE, and standard electroencephalographic evaluations were performed in these patients. Results: In contrast to periodic epileptiform discharges, erroneous SE and RE values in the patients’ EEGs were associated with the presence of polyspikes (PS) and rhythmic polyspikes (PSR), which were more likely to indicate toxic depth rather than false emergence from anaesthesia with no changes in the FiAA, FeAA, and MAC of sevoflurane. Conclusion: Calculated RE and SE values may be misleading during VIGA when EPs are present in patients’ EEGs. During VIGA with sevoflurane, we recommend monitoring raw EEG data in scientific studies to correlate it with potentially erroneous RE and SE values and the end-tidal concentration of sevoflurane in everyday clinical practice, when monitoring raw EEG is not available, because they can mislead anaesthesiologists to reduce sevoflurane levels in the ventilation gas and result in unintentional true emergence from anaesthesia. Further studies are required to investigate the behaviour of EEG-based indices during rapid changes in sevoflurane concentrations at different stages of VIGA and the influence of polyspikes and rhythmic polyspikes on the transformation of EEG signals into a digital form.  General anaesthesia is sometimes associated with unwelcome events. Both volatile induction of general anaesthesia (VIGA) with sevoflurane and intravenous induction of general anaesthesia with propofol may induce seizure-like movements or seizures accompanied by haemodynamic instability ,2, whichIntraoperative awareness during general anaesthesia is a distressing complication with the potential for long-term psychological consequences , and theElectroencephalographic (EEG) signals are derived from a frontal sensor, which is quickly and easily applied to the patient’s forehead and does not entail complex preoperational preparations. The EEG signal is then transformed to a digital score between 0 and 100 visible on the anaesthetic monitor to control the hypnotic component of general anaesthesia: values over 80 indicate an awake state; between 60 and 80, sedation; between 40 and 60, a level of unconsciousness suitable for surgery; between 30 and 40, too deep anaesthesia; and under 30, overdose of anaesthetic ,20. ThisBIS values during the presence of EPs on the EEG  under GAThe abovementioned results suggest that EEG-based indices do not always provide a reliable measure of anaesthetic depth; hence, anaesthesiologists should not rely exclusively on the BIS and SE readings when assessing depth of anaesthesia. We had previously conducted a comparative, prospective study to assess the influence of VIGA with sevoflurane using two different techniques and intravenous induction of general anaesthesia using propofol on the possible presence of EPs . This stPatients who were scheduled to undergo elective orthopaedic knee surgery under combined general and regional anaesthesia in the Clinic of Orthopaedics at St. Barbara’s Memorial Hospital no. 5 in Sosnowiec, Poland, and met the inclusion criteria were asked to participate in the study. Sixty adult patients under 70 years of age with American Society of Anaesthesiologists (ASA) scores of I-II were enrolled after obtaining written informed consent. Ethical approval for this study (NN-6501-196/06) was provided by the Ethical Committee of Medical University of Silesia on December 19, 2006; the study was registered in the Clinical Trial Registry .The exclusion criteria were as follows: a history of epilepsy, medical treatment that might interfere with the EEG assessments , or a neurological disease or a neurosurgical operation that would impair EEG or entropy EEG monitoring; pregnancy; drug or alcohol abuse; a history of pulmonary disease; or signs predicting difficult mask ventilation or intubation. To exclude any pre-existing epileptic EEG patterns, standard 30 min initial EEG recordings were performed for all patients participating in the study. The initial EEG was taken in a dark quiet room for 5 min as a baseline, followed by three eye-opening and closing sequences of 10 s each and photostimulation lasting 10 min . Then, another baseline reading was obtained, and the patients were asked to achieve a state of hyperventilation by taking 20 forceful breathes per minute for five minutes. Finally, another baseline reading was obtained. Only patients without EPs in their EEGs were included into final analysis (Prof. Ville Jäntti). The fifty-five patients included in the study were randomly assigned to one of the three groups  using blind random allocation via sealed envelopes. 2 was maintained at 35–45 mmHg since hyperventilation may trigger epileptiform activity before and during the induction of general anaesthesia. After LMA placement, before the operation started, sevoflurane concentration was maintained at the level of 1 MAC. Before the induction of anaesthesia, none of the patients received any medication on the day of surgery. Directly before surgery, the patients were preoxygenated for 5 min with 100% oxygen and intravenously administered 10 mL/kg per body weight of Ringer solution. The patients in the VIMA group were anaesthetised with sevoflurane using the increasing concentrations technique. The patient was breathing spontaneously via a face mask and the sevoflurane concentration in the inhaled gas was doubled every 10 breaths, starting from 0.3 vol% and proceeding in the sequence 0.3–0.6–1.2–2.4–4.8–8 vol.% until a minimal alveolar concentration (MAC) of 2 was obtained in the exhalation gas. The patients in the VCRII group were anaesthetised with 8% sevoflurane and 92% oxygen using the vital capacity technique. The anaesthetic circuit was prefilled with 8% sevoflurane, and a face mask was applied to the patient’s face to allow them to breathe spontaneously. In group C, the patients were preoxygenated with 100% oxygen and received propofol intravenously at a single dose of 2.5 mg/kg of body weight with subsequent propofol infusion at an infusion speed of 4 mg/kg body weight/hour. Assisted ventilation was initiated in all groups after loss of consciousness (LOC) when hypoventilation appeared and the ciliary reflex disappeared. When the minimum MAC of 2% was reached for sevoflurane in patients from the VIMA and VCRII groups or 5 min after the bolus of propofol with subsequent propofol infusion in patients in the PROP group, the patients in all groups were paralysed with a standard intravenous dose of 0.08–0.1 mg/kg cisatracurium , and a laryngeal mask (LMA) was applied after 45 s. CO2), fraction of inspired oxygen in the gas mixture (FiO2), facial electromyography (fEMG), fraction of inspired sevoflurane (FiAA), fraction of expired sevoflurane (FeAA), exhaled carbon dioxide concentration (EtCO2), minimal alveolar concentration of sevoflurane (MAC), and depth of anaesthesia using response entropy (RE) and state entropy (SE)  [Throughout anaesthesia induction and surgery, standard monitoring procedures were utilised and close attention was paid to vital parameters such as non-invasive arterial pressure (BP), heart rate (HR), standard electrocardiography (ECG) II, arterial blood saturation (SaOFinland) .2 cup electrodes (Spes Medica) attached to the scalp with EC2 Electrode Cream (Grass Technologies). The electrodes  were positioned on both temporal bones lateral to the eyes, on both mastoid bones, on Fp1, Fp2  and Fpz, and the ground electrode was positioned in the centre of the forehead between the eyebrows. Four-channel EEG was recorded using the following electrode pairs: Fp1—left mastoid, Fp2—right mastoid, Fpz—left temporal, and Fpz—right temporal [The data were collected and digitised at 10 s intervals using the S5/Collect (Datex-Ohmeda Division) and Bispectrum Analyzer for BIS (BSA for BIS version 3.22B2 for A-2000—S. Hagihira) installed on a notebook computer (Fujitsu-Siemens Amilo Pi1515). Four EEG channels were recorded using Ag/AgCltemporal  . Since NMBDs are reported to decrease the BIS value , the momp < 0.05 .Statistical analyses were performed using MS Excel, STATISTICA 12, Stat Soft , and R 3.3.2, GNU General Public License. The measured data were characterized using the mean and standard deviation (X ± SD) as well as medians with interquartile range (M IQR). Normality of distribution was checked with the Shapiro–Wilk W test. The significance of differences between means was tested using ANOVA for multiple groups, and for skewed distributions, their compatibility in groups was examined using the Kruskal–Wallis test by ranks. Comparison of average values of tested parameters at different stages was performed via one-way analysis of variance (ANOVA) for repeated measurements and the Friedman Rank Sum test. Two-way analysis of variance (ANOVA) for repeated measures and the nonparametric aligned rank test for repeated measures were conducted in two-factor analysis, with the group and the stage of the study serving as factors. For nominal data, we used percentages. Relationships between nominal variables were verified by the Fisher’s exact test for n by m tables. Evaluation of the relationship between two continuous variables was performed using the Spearman correlation coefficient R’ with an appropriate test of significance. Statistical significance was set at the level p = 0.664), body weight , height , and female-to-male participant ratios . In the VCRII group, EPs were registered in 7 (21.9%) patients, while in the VIMA group, EPs were registered in 10 (31.3%) patients. Out of the total of 60 patients allocated in the current study, 20 patients from the VIMA group, 20 from the VCRII group, and 20 from the PROP group were included in further analyses. Four patients each from the VIMA and VCRII groups were excluded due to possible artefacts in their EEG signals . Patients in the VIMA, VCRII, and PROP groups showed no significant differences in terms of age . In the VCRII group, EPs were registered in 7 (21.9%) patients, while in the VIMA group, EPs were registered in 10 (31.3%) patients. The patients’ parameters were analysed during the following six stages of VIGA: stage 1, onset of VIGA; stage 2, after LOC defined as absence of ciliary reflex; stage 3, 10 s before appearance of the very first EP; stage 4, the first 10 s of the very first EP; stage 5, when the highest RE and SE values appeared in the presence of EPs; and stage 6, when the highest RE and SE values appeared at least 10 s after disappearance of EPs. The time intervals from the start of VIGA (stage 1) to the very first EP in the EEG (stage 4) were significantly different between the VIMA and VCRII groups .The patients’ parameters were analysed during the following six stages of VIGA: stage 1, onset of VIGA; stage 2, after LOC defined as absence of ciliary reflex; stage 3, 10 s before appearance of the very first EP; stage 4, the first 10 s of the very first EP; stage 5, when the highest RE and SE values appeared in the presence of EPs; and stage 6, when the highest RE and SE values appeared at least 10 s after disappearance of EPs. The time intervals from the start of VIGA (stage 1) to the very first EP in the EEG (stage 4) were significantly different between the VIMA and VCRII groups  before the induction of anaesthesia (stage 1); (2) after loss of consciousness (LOC) (stage 2); (3) before the presence of the first EP in the EEGs in the VIMA-EP and VCRII-EP groups, and the mean time to the potential presence of the first EP in the EEGs in the VIMA-nEP and VCRII-nEP groups (stage 3); (4) during the presence of the first EP in the EEGs in the VIMA-EP and VCRII-EP groups and during the mean time interval to the potential presence of the first EP in the EEGs in the VIMA-nEP and VCRII-nEP groups (stage 4); (5) during the presence of the highest RE and SE values associated with the first EP in the EEGs in the VIMA-EP and VCRII-EP groups and the highest RE and SE values in the EEGs in the VIMA-nEP and VCRII-nEP groups; and (6) during the presence of the highest RE and SE values associated with the disappearance of EPs in the EEGs in the VIMA-EP and VCRII-EP groups and the highest RE and SE values in the EEGs in the VIMA-nEP and VCRII-nEP groups . The RE The influence of induction on the levels of these parameters at individual stages in the study is presented in There was a significant decline in the SAP, MAP, fEMG, SE, and RE values in patients induced using the VCRII technique with no EPs (VCRII-nEP group) in the final stages of the study . The lower fEMG values at the fourth and fifth stages were noted in patients induced using the VIMA technique with EPs in their EEGs, a significant increase in the value at the final stage in the case of SE and RE was observed . We observed normative values of SE and RE (40–60) in less than 10% of anaesthetised patients regardless of the analysed stage of VIGA. A significantly higher percentage of patients who possibly recovered from anaesthesia  among the patients induced using the VCRII technique was observed at the third stage, while most of those in deep anaesthesia at this stage belonged to the VIMA group. For all patients induced with the VCRII technique, we noted deep anaesthesia (based on the parameter RE) at the fifth stage of the study . Among patients induced using the VCRII technique and showing EPs (VCRII-EP group), significantly higher values of SE and RE were associated with higher values of HR. Significant negative correlations between SE and SAP, DAP and MAP were observed for the patients who were induced with the VCRII technique and showed no EPs (VCRII-nEP). In the case of RE, correlation parameters were related to DAP and MAP. Negative correlation of RE with fiAA values was noted in patents induced with VCRII technique and showing the development of EPs (VCRII-EP group) . During the presence of EPs, PS and PSR that may falsely indicate intraoperative awareness were present regardless of the anaesthetic regimen used, whereas PEDs appeared mainly during SE values that may falsely indicate deep anaesthesia . Interestingly, in one male patient aged 41 years  induced with sevoflurane using the VIMA technique, before the appearance of EPs (stage 3), the values of SE and RE were 60 and 79, respectively . When PS appeared in his EEG recordings, during the first 10 s, the SE and RE values decreased to 45 and 59, respectively  and subsequently increased to 80 and 94, respectively, 90 s later , indicating false recovery from anaesthesia, whereas the depth of anaesthesia measured by clinical parameters did not change during the transition from stages 3 to 4 and 5. Epileptiform activity during the abovementioned increase in RE and SE is presented in Surprisingly, in another male patient aged 23 years, 140 s after induction was started using VIGA with sevoflurane (stage 1) and 50 s after LOC (stage 2), we observed convulsions following tonic-clonic generalized seizures. Further EEG pattern analysis revealed the development of spiky activity. In this case, evident epileptiform activity in the patient’s EEG recordings accompanied by clinically manifested tonic-clonic seizures that did not result in a rapid increase of RE and SE values, because the observed values of RE and SE were 7–18 and 7–15, respectively, indicating toxic concentrations of sevoflurane in the ventilation gas, whereas at the time of the lowest RE and SE values , the FeAA, FiAA, and MAC values were 4.52, 7.62, and 2.20, respectively .In our study, induction of VIGA with sevoflurane resulted in the presence of EPs in patients’ EEGs at a similar rate to previous studies concerning the presence of EPs during GA with sevoflurane ,7,26,27.To our knowledge, the problems associated with aberrant values of EEG-based indices in the presence of EPs have been quite well recognized in terms of BIS ,26,27, bVisual inspection of the EEG signals for detection of normal, interictal, and ictal activities is a challenging task due to the huge volumes of EEG segments that have to be studied offline or the speed of EEG pattern changes when monitoring online during GA. Therefore, attempts have been made to utilise non-linear methods to study EEG signals for automatic monitoring of epileptic activities. The ultimate goal of clinical anaesthesiologists is to develop a computerized technique that can classify all the three classes of EEG segments , and the need for such software directs current research to investigate the possibility of application of automatic seizure monitoring in the near future, which could provide a real-time EEG-based brain monitoring system for epileptic seizure prediction .The exemplary studies in this field investigating either the utility of observance of distribution entropy values  sample aIn our study on the fifth stage after induction using the VIMA technique, where EPs were present, RE and SE values were significantly higher than those in patients who showed no EPs in their EEGs at the same stage, similar to the findings obtained by Li P et al. . At the third stage of the study analysis (before the appearance of EPs in patients’ EEGs), higher values of SE and RE were observed in the group of patients with EPs in their EEGs after induction using the VCRII technique (VCRII-nEP group), in comparison to stages 4 (first 10 s of the presence of EPs) and 5, when RE and SE values where the highest in the presence of EPs, similar to findings of Lu WY et al. . Rapid decrease in RE and SE in the VCRII-EP group during the transition from stage 3 to 4 in the current study analysis, and contrary to our observation in the VIMA-EP group, where during the transition from stage 3 to 4, such reductions were not present were also observed by Weng WC et al. . Globally, at the third stage of VIGA with the potential presence of EPs, 13 patients presented with SE values indicating recovery from GA, 5 patients showed SE values indicating potential recovery from GA with recall, whereas no patient showed values indicating a surgical level of anaesthesia, and 13 patients showed values indicating almost toxic concentrations of sevoflurane in the ventilation gas. However, no differences in FiAA, FeAA, and MAC were observed at this time point. At the fourth stage in 3 patients from the VCRII group, after appearance of EPs, both RE and SE values indicated potential recovery from GA, but in patients with no EPs in their EEGs, such values were not observed. Therefore, we conclude that the appearance of EPs may elevate RE and SE values falsely, falsely indicating insufficient concentration of sevoflurane in ventilation gas. Interestingly, in 24 patients, the SE values represented almost toxic concentrations of sevoflurane that almost doubled during the transition from stage 3 to 4 with no corresponding changes in FiAA, FeAA and MAC. At the fifth stage, in one patient from the VIMA group who showed EPs, both RE and SE values elevated to a level corresponding to recovery from GA. For almost 30 patients, these values stayed < 40, indicating too deep anaesthesia, with no statistically significant changes in FiAA, FeAA and MAC during the transition from stage 4 to 5; this was identical to our observations for a patient presenting with generalised tonic-clonic seizures whose RE and SE values were <20 during spiky activity in his EEG recordings despite moderate FiAA, FeAA, and MAC at this time. Monitors showing EEG-based indices, which evaluate the hypnotic component of anaesthesia by analysing patients’ EEGs, proved helpful in decreasing the number of incidences of intraoperative awareness with recall . On the We observed that a significantly higher percentage of patients falsely recovered from anaesthesia  among the patients induced using VCRII technique at the third stage (before EPs appeared), while among those in deep anaesthesia, most patients were induced using the VIMA technique. We suppose that abovementioned difference might have been caused by a shift in transformation of the EEG signal detected by a frontal sensor into RE and SE value in patients who underwent VIGA with the VCRII technique, which is very fast. Conversely, such correlations were not observed in the VIMA group since induction of VIGA in these patients was relatively slow, so the shift in transformation of EEG signal into RE and SE value might have been of no clinical relevance.In our study, EPs appeared on average after 209 ± 195 s after the onset of VIGA in patients anesthetized using VCRII. In three patients from the VCRII group, RE and SE values reflected potential false recovery from anaesthesia during the presence of EPs in their EEGs, whereas in patients anesthetized using the same technique who showed no EPs in their EEG at the fourth stage (VCRII-nEP group), all patients had RE values < 40 and SE values < 30, possibly falsely indicating toxic concentrations of sevoflurane in the ventilation gas. At the fifth stage, the SE values decreased below 40, mainly falsely representing a toxic level of anaesthesia (SE < 30 in 26 patients) in the VCRII-EP group despite the continuous presence of PS-type EPs. Surprisingly, in one patient anesthetized using the VIMA technique, at fifth stage of VIGA, SE and RE increased to >80 during the presence of PS-type EPs, possibly falsely indicating recovery from anaesthesia despite the steadily high concentration of volatile anaesthetic in ventilation gas at stages 3, 4 and 5. In this case, a potential shift in transformation of the EEG signal into a digital form is rather not likely to explain the sudden increase in RE and SE values, as at the fourth stage their indicated surgical level of anaesthesia  and concentration of sevoflurane in ventilation gas was not higher than that in the previous stages. Since the RE and SE values in a huge majority of patients at stage 5 reflect toxic concentrations of sevoflurane, regardless of technique used and despite the absence of changes in FeAA, FiAA, and MAC of volatile anaesthetics, further studies are required to investigate if RE and SE values may not falsely show too deep anaesthesia than it really is. Similar observations were obtained Laitio RM et al.  who obseIf SE values unreliably show too deep a level of anaesthesia as a result of the contribution of high-amplitude delta activity, it may possibly be hazardous for the patient because the anaesthesiologist could decrease the concentration of sevoflurane in ventilation gas and unintentionally awake the patient, if depth of anaesthesia is blindly guided based on observance of RE and SE values only. On the other hand, the time needed for transformation of EEG signal derived from frontal sensor might be the explanation for the fact that the concentration of end-expiratory sevoflurane increases faster during VIGA than the values of RE and SE decrease, and there may be a time delay in the reflection of the actual depth of anaesthesia in a huge majority of cases regardless of the appearance of EPs in patients EEGs, especially when the VCRII technique is utilised. Therefore, we suggest that further studies are needed to investigate if RE and SE reliably reflect the actual depth of anaesthesia during rapid changes of sevoflurane concentration in the end-expiratory gas by comparing values of different EEG-based indices that are popularly used to monitor depth of anaesthesia, and comparing them with EEG patterns, especially during the presence of PS-type EPs in patients’ EEGs. On the margin, in all patients who had EPs in their EEGs, we observed patterns of the PED type, but they tended to appear after NMBD administration, so we did not perform analysis afterwards. NMBD are known to influence values displayed by depth of anaesthesia monitors, which is well-recognised in the case of BIS scores ,50,51,52In the end, since a lag in time is probably needed for transformation of the EEG signal recorded from a frontal sensor into a digital form displayed on the monitor, this factor could constitute the main limitation of the present study’s assessments, especially concerning VCRII group. Therefore, from a more practical point of view, we recommend on-line observation of variations in RE and SE values as well as FeAA, FiAA, and MAC of volatile anaesthetics, especially during the VCRII technique, because their verification against raw online EEG patterns in everyday practise is not available. Relying only on RE and SE values to guide the depth of anaesthesia may possibly lead to making a wrong decision resulting with either too deep anaesthesia or intraoperative awareness with recall. During different stages of VIGA with sevoflurane, RE and SE values may vary, showing a wide variety of values from >80, which may falsely represent recovery from anaesthesia during presence of EPs in patients’ EEGs, to <30, which may falsely indicate a toxic concentration of sevoflurane, regardless of the steady concentration of sevoflurane in the ventilation gas. The values of SE and RE during the presence of EPs in patient’s EEGs indicating false recovery from anaesthesia were associated with PS, whereas the presence of PEDs did not result in the presence of aberrant RE and SE values. RE and SE values during the presence of EPs are more likely to indicate toxic depth of anaesthesia rather than false recovery from anaesthesia with no change in FiAA, FeAA of sevoflurane, and MAC values between stages. Abovementioned aberrant RE and SE values could mislead anaesthesiologists into changing sevoflurane levels in the ventilation gas and either possibly lead to unintentional either recovery from anaesthesia or administering toxic concentration of sevoflurane, if depth of VIGA with sevoflurane is blindly guided based on observance of RE and SE values only. Further studies are required to investigate the behaviour of EEG-based indices during rapid changes in sevoflurane concentration at different stages of general anaesthesia and the influence of EPs on the transformation of EEG signals into a digital form. Therefore, during VIGA with sevoflurane, we recommend monitoring raw EEG in scientific studies to correlate it with the values of RE and SE and the end-tidal concentration of sevoflurane in everyday clinical practice, when monitoring raw EEG is not available.


When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures  RNA structure is a jack-of-all-trades. Besides being crucial to the function of structural non-coding RNAs (ncRNAs), RNA structural elements are now widely recognized as key players in almost any essential biological process, ranging from transcriptional and post-transcriptional control of gene expression, to catalysis and sensing of environmental stimuli such as metabolite and temperature changes .n nucleotides-long RNA can theoretically fold in up to 1.8n distinct conformations  (A widely used approach to define the putative structure of an RNA based on its sequence relies on predicting its minimum free energy (MFE) structure, the theoretically most stable structure, based on a set of experimentally-determined thermodynamic parameters known as r rules) . Unfortur rules) . This isr rules) . The accr rules) .in vivo, while the majority of them are only suitable for in vitro and ex vivo RNA structure probing experiments. Two classes of compounds are mainly used to date. On the one hand, nucleobase-specific probes, such as dimethyl sulfate (DMS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), can be respectively used to probe adenine (A) and cytosine (C), or guanine (G) and uracil (U) bases  bases ,14. On tU) bases , as theyU) bases ,17, withU) bases ,19.Typical readout of RNA probing experiments is the detection of reverse transcription (RT) drop-off events induced by the probing reagent-modified RNA bases . More rein vivo RNA probing by synthesizing six new SHAPE reagents and by assaying their ability to probe in vivo Gram-positives, Gram-negatives and mammalian cells. One of these compounds, 2-aminopyridine-3-carboxylic acid imidazolide (2A3), showed both increased reactivity with RNA and higher permeability to biological membranes, resulting in a significantly higher signal-to-noise ratio when compared to NAI. Importantly, when used to perform experimentally-informed prediction of in vivo RNA structures, 2A3 produced markedly more accurate predictions than NAI.While the use of SHAPE compounds over nucleobase-specific probes has in theory a considerable advantage, as they enable the unbiased probing of all four RNA bases, reagents such as DMS still exhibit a higher signal-to-noise ratio in MaP experiments. To this end, we here sought to optimize Ka close to (or slightly above) 7. The pKa values in water were obtained (where possible) using the DataWarrior tool  and added to D2O to achieve quantifiable concentrations. Samples were incubated at 25°C or 37°C and kinetic 1H NMR scanning was performed. The disappearance of product resonances and reappearance of starting materials (correlating to hydrolysis) was monitored as previously described . 2 ml aliquots were collected by centrifugation at 1000 × g (4°C) for 5 min. Cell pellets were resuspended in 1 ml of Resuspension Buffer , and lysozyme was added to a final concentration of 100 μg/ml. After incubation at 22°C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 × g (4°C) for 5 min. Pellets were resuspended in 120 μl Protoplast Lysis Buffer , supplemented with 0.2 μg/μl Proteinase K. Samples were incubated for 5 min at 22°C and for 5 min on ice. Sample was then extracted 2 times with phenol:chloroform:isoamyl alcohol , and once with chloroform. 20 U SUPERase•In™ RNase Inhibitor  were then added and RNA was equilibrated at 37°C for 20 min prior to probing.Deproteinized escribed , with miE. coli rRNAs in RNA folding buffer, pre-equilibrated at 37°C for 20 min, were mixed with 20 μl of each SHAPE compound . RNA was then allowed to react at 37°C for 15 min, with moderate shaking, after which 200 μl of 1 M DTT were added, to quench the reaction. Samples were then vortexed briefly and 1 ml of ice-cold QIAzol Lysis Reagent  was added to each sample, followed by extensive vortexing.180 μl of deproteinized E. coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000 × g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 100 μl of Buffer A , supplemented with 5 U SUPERase•In™ RNase Inhibitor, by vigorously vortexing for 30 s. 25 μl of Buffer B , supplemented with 100 μg/ml final lysozyme, were then added, followed by 30 s vortexing. Samples were incubated 1 min at room temperature, after which 125 μl of Buffer C  were added to lyse protoplasts. Samples were then incubated at room temperature for 5 min, after which 100 μl of lysate were transferred to 1 ml ice-cold QIAzol and thoroughly mixed by vortexing.A single colony of Bacillus subtilis 168 was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000× g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 200 μl TE Buffer , supplemented with 1.1% final SDS. 0.25 g glass beads were then added and samples were beated in a Mini-Beadbeater-24 (Glen Mills) for 1 min. Samples were then incubated on ice for 1 min, after which beating was repeated once. 1 ml ice-cold QIAzol was then added, followed by vigorous vortexing. Samples were then incubated at 70°C for 20 min, to allow complete cell lysis.A single colony of d-glucose), supplemented with 10% heat-inactivated FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 U/ml penicillin and 25 μg/ml streptomycin, at 37°C (5% CO2), to ∼75% confluence. Cells were then covered with a thin layer of trypsin–EDTA (0.25%) solution, incubated at room temperature for 1 min, then dissociated by pipetting up and down with complete medium. Cells were centrifuged at 180 × g for 1 min and medium discarded. Cells were then resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by gentle mixing by tapping the tube. Cells were allowed to react at 37°C for 15 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 10 000 × g for 1 min (4°C) and supernatant discarded. Cells pellets were lysed by direct addition of 1 ml ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates.HEK293 cells were grown in DMEM (4.5 g/l g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 ml tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column  and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Besides samples treated with I6, all other samples appeared to be perfectly intact. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase  at 37°C for 20 min.After samples have been collected in 1 ml QIAzol, 200 μl of chloroform was added, followed by vigorous vortexing for 15 s. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12 500 × 2], then purified with NucleoMag NGS Clean-up and Size Select beads , supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 μl (for SSII and HIV RT) or 2.5 μl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 μl 20 μM random hexamers and either 0.25 μl (for SSII) or 0.5 μl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 μl. For HIV RT, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT . For TGIRT-III, reaction was supplemented with 1 μl 5× RT Buffer , 0.25 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT . For SSII, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 0.25 μl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT . Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III) or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 μg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 μl of a 1:2 dilution of protease inhibitor cocktail  in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module . Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® , following manufacturer instructions.Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer {3})’. For each hairpin-loop, we then calculated the median reactivity on the first and last two bases of the loop and the median reactivity on the three bases of the stem preceding and following the loop. The signal-to-noise value was then calculated as the ratio between the median reactivity across all the loops and the median reactivity across all the stems.E. coli cells, under conditions previously shown to preserve the native RNA folding  and HIV RT (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) adducts (HIV RT). Analysis of the distribution of per-base mutation frequencies across 16S and 23S rRNAs showed that, while TGIRT-III has lower background mutation frequencies compared to SSII (median DMSO control: 0.75 × 10−3 for TGIRT-III versus 2.19 × 10−3 for SSII) as previously reported , we sought to find the best reverse transcription conditions. To this end, we extracted deproteinized total RNA from  folding ,23,33 and HIV RT ,24,34. Treported , SSII shreported . Accordie models  and inspe models . Altogetin vivo probing experiments); (ii) high signal over background ; (iii) easy and safe to synthesize. The two best-characterized in vivo SHAPE reagents to date are NAI and FAI. These compounds can be easily synthesized via the carbonyldiimidazole (CDI)-mediated activation of carboxylic acids, resulting in the corresponding carboxylic acid imidazolide, an activated carbonyl, that can acylate nucleophiles present in the system, especially 2′ hydroxyl groups of RNA. We decided to adopt the same strategy, trying to optimize the acylating group. To this end, we selected seven commercially-available carboxylic acids, preferring those containing nitrogen groups or for which the putative adduct-forming group had a pKa in water close to (or slightly above) 7  permeable to biological membranes  as it was too reactive, hence resulting in very fast RNA degradation (data not shown). When looking at overall distributions of mutation rates, indoline-5-carboxylic acid imidazolide (I5) and 1-methylimidazole-4-carboxylic acid imidazolide (1M4) showed a lower reactivity towards RNA compared to NAI . Oppositely, the other four compounds, nicotinic acid imidazolide (NIC), benzotriazole-5-carboxylic acid imidazolide (B5), 6-aminopyridine-3-carboxylic acid imidazolide (6A3) and 2-aminopyridine-3-carboxylic acid imidazolide (2A3), all showed increased reactivity compared to NAI , with 2A3 having ∼2.4X higher mutation frequencies than NAI , independently of DMSO background signal subtraction , independently of the probed cell type, partially followed by B5 in B. subtilis and NIC in HEK293 cells.At first, we looked again into the distribution of mutation frequencies for the different reagents. Unexpectedly, while the observed distributions for HEK293 cells approximately mirrored those observed on ied RNAs , we obsee Figure . On the ex vivo-probed RNAs, ROC curves built with respect to unpaired versus paired residues from E. coli, B. subtilis and H. sapiens rRNAs  revealed a marked difference in sensitivity/specificity of NAI compared to 2A3, with 2A3 showing up to 17–20% increased discrimination of unpaired versus paired residues, independently of DMSO background signal subtraction  with respect to NAI , independently of the examined cell type  and the intercept (b). As these parameters depend on the experimental setup and on the employed reagent, we first used the ViennaRNA Package 2.0  and positive predictive value  for both 16S and 23S rRNAs. Notably, grid search already revealed that the accuracy of 2A3-driven predictions was largely independent of the employed slope/intercept pair and that, even the most accurate NAI-driven prediction  was ∼10% less accurate than most 2A3-driven predictions, independently of the used m/b pair and ∼18% less accurate that the top-scoring 2A3-driven prediction . As an example, although incorporation of NAI data slightly increased the accuracy with respect to the unconstrained prediction , the NAI-guided prediction resulted in the misfolding of the 3′ domain of the 16S rRNA, while the 2A3-guided prediction correctly recapitulated most of the known base-pairs from the accepted reference structure . This unwanted side product increased over time, eventually reaching a maximum of ∼90% as compared to 2A3 in solution during an overnight reaction in a sealed vessel. The formation of 2A3 proceeds rapidly as CDI is added to the 2-aminonicotinic acid. The conversion of 2A3 to 3AIA then proceeds at room temperature in the crude reaction mixture. The unwanted side reaction proceeds slowly and can be minimized with vigorous stirring under a constant flow of inert gas.in vivo SHAPE probe, we obtained commercially-available pure 3AIA  and used it to probe living E. coli cells. Notably, 3AIA showed modification frequencies indistinguishable from those of the DMSO control in vivo  showed partial improvements with respect to NAI. Surprisingly, when tested in vivo, 2A3 significantly outperformed NAI and all the other tested reagents under all the analyzed conditions, with the most striking differences being observed in bacterial cells. Indeed, for both E. coli and B. subtilis, several regions of the ribosome resulted to be blind to probing by NAI and most of the other tested reagents, while they were readily probed by 2A3. In this context, 2A3 better captured local nucleotide dynamics, as confirmed by in depth analysis of available ribosome 3D structures. This reveals that, besides showing a higher ability to react with RNA, 2A3 has also a greater capacity to permeate biological membranes. It is conceivable that the peptidoglycan of the bacterial cell wall might represent the main limitation when it comes to in vivo SHAPE probing. Indeed, although 2A3 is able to probe both gram-positive and gram-negative bacteria, we observed higher efficiencies in gram-negatives, suggesting that the thicker peptidoglycan of gram-positives limits the permeability to the SHAPE compound. The presence of an amine rather than a methyl group at position C2, that differentiates 2A3 from NAI, might explain this increased permeability, in agreement with a recent study showing that small molecules containing amine groups are more likely to permeate and accumulate into bacteria  even if providing information on roughly only a half of RNA bases.Furthermore, we showed that the higher signal-to-noise ratio achieved by 2A3 results in a significantly higher RNA structure prediction accuracy when performing 2A3-informed RNA folding. It is worth noticing that 2A3, as opposed to NAI, outperforms probing with dimethyl sulfate (DMS) as well. Indeed, probing with DMS usually shows a higher signal-to-noise ratio with respect to NAI, hence resulting in more accurate experimentally-informed RNA structure predictions . It may be that 2A3 falls in a ‘sweet’ spot for reactivity compared to the other tested electrophiles both in vivo RNA structurome.In summary, 2A3 represents a concrete and significant advance in our ability to interrogate RNA structures in living cells. We can anticipate that, being able to readily probe even regions of ribonucleoprotein complexes that are normally hidden to other compounds, 2A3 will rapidly overtake conventional SHAPE reagents, revealing new and previously unobserved features of the ex vivo probing of E. coli RNA with DMSO and NAI has been previously deposited under the accession GSE151327, as part of another study. Additional processed files are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php.SHAPE-MaP data has been deposited to the Gene Expression Omnibus (GEO) database, under the accession GSE154563. Data for gkaa1255_Supplemental_FileClick here for additional data file.


Experimental data from in vivo tests showed that test compounds 3a–3c, 3f, and 3g (50 mg/kg) significantly prolonged reaction times of animals in tail-clip and hot-plate tests compared to the controls, indicating that these compounds possess centrally mediated antinociceptive activities. Furthermore, these compounds reduced the number of writhing behaviors in the acetic acid-induced writhing tests, showing that the compounds also possess peripheral antinociceptive activity. In the mechanistic studies, naloxone pre-treatments abolished the antinociceptive activities of compounds 3a–3c, 3f, and 3g, indicating that opioidergic mechanisms were involved in their antinociceptive effects. Molecular docking studies demonstrating significant interactions between the active compounds and µ- and δ-opioid receptor proteins supported the pharmacological findings. This study is the first showing that molecules designed to bear thiazole and piperazine moieties together on their structure exert centrally and peripherally mediated antinociceptive effects by activating the opioid system.In this study, we aimed to design and synthesize novel molecules carrying both the thiazole and piperazine rings in their structures and to investigate their antinociceptive activity. Targeted compounds were obtained by reacting thiosemicarbazide derivative and appropriate 2-bromoacetophenone in ethanol. The structures of the obtained compounds were determined using data from various spectroscopic methods (IR,  Pain is a health problem affecting the quality of life of patients due to its prevalence and accompanying disabilities. The pharmacological agents used in the treatment of pain include nonsteroidal anti-inflammatory drugs (NSAID), opioid analgesics, and analgesic adjuvants  . AlthougNitrogen- and sulfur-containing heterocycles are frequently used in drug synthesis. Thiazole, a five-membered ring system carrying three carbons, one nitrogen, and one sulfur atom, is one such structure. It has been reported that thiazole-bearing compounds have some central nervous system (CNS)-related effects, such as anti-schizophrenic , anti-pa2 adrenergic, adenosinergic, and D2/3 dopaminergic receptors +: 442.09.Yield: 85%, Rf = 0.57, M.P. = 210–212 °C, FTIR : 3294 (-NH), 2856 (C-H), 817. 1H-NMR : δ = 2.32 , 2.93 , 3.23–3.26 , 3.33–3.36 , 7.03 , 7.19–7.22 , 7.53 , 7.74 , 7.94 , 11.93 . 13C-NMR : δ = 21.27, 34.32, 45.59, 47.77, 102.74, 115.93, 125.67, 125.91, 127.89, 128.75, 129.62, 132.56, 137.19, 141.98, 151.45, 168.69. ESI-MS (m/z): [M + H]+: 456.11.Yield: 82%, Rf = 0.68, M.P. = 226–230 °C, FTIR : 3294 (N-H), 2845 (C-H), 819. 1H-NMR : δ = 2.93 , 3.23–3.25 , 3.33–3.36 , 3.78 , 6.96 , 7.02 , 7.10 , 7.52 , 7.78 , 7.93 , 11.93 . 13C-NMR : δ = 34.31, 45.59, 47.77, 55.55, 101.51, 114.03, 114.40, 115.93, 125.69, 127.28, 127.88, 128.07, 141.96, 151.44, 159.20, 168.68. ESI-MS (m/z): [M + H]+: 472.10.Yield: 87%, Rf = 0.77, M.P. = 175–178 °C, FTIR : 3292 (N-H), 2845 (C-H), 2222 (C≡N), 817. 1H-NMR : δ = 2.92 , 3.23–3.26 , 3.33–3.37 , 7.02 , 7.53 , 7.59 , 7.86 , 7.96 , 8.03 , 12.01 . 13C-NMR : δ = 34.42, 45.58, 47.76, 107.58, 109.98, 115.90, 119.47, 125.51, 126.56, 127.99, 133.13, 139.32, 142.57, 149.27, 151.55, 169.13. ESI-MS (m/z): [M + H]+: 467.09.Yield: 89%, Rf = 0.34, M.P. = 237–239 °C, FTIR : 3305 (N-H), 2845 (C-H), 1504, 1334 (NO2), 819. 1H-NMR : δ = 2.92 , 3.23–3.26 , 3.34–3.37 , 7.02 , 7.53 , 7.67 , 7.96 , 8.10 , 8.27 , 12.05 . 13C-NMR : δ = 34.42, 45.58, 47.75, 108.60, 115.89, 124.55, 125.47, 126.77, 128.01, 141.22, 142.66, 146.63, 148.97, 151.57, 169.22. ESI-MS (m/z): [M + H]+: 487.08.Yield: 88%, Rf = 0.79, M.P. = 220–223 °C, FTIR : 3344 (N-H), 2841 (C-H), 817. 1H-NMR : δ = 2.92 , 3.23–3.25 , 3.33–3.35 , 6.98 , 7.22–7.27 , 7.49–7.52 , 7.64 , 7.83–7.86 , 7.96 , 11.24 . 13C-NMR : δ =34.42, 45.58, 47.64, 47.79, 103.41, 115.75 (J = 26.08 Hz), 125.20, 125.64, 127.92, 128.99, 142.19, 143.07, 151.49, 151.85, 162.04 (J = 244.24 Hz), 168.90, 177.84. ESI-MS (m/z): [M + H]+: 460.08.Yield: 79%, Rf = 0.31, M.P. = 190–193 °C, FTIR : 3300 (N-H), 2835 (C-H), 817. 1H-NMR : δ = 2.93 , 3.23–3.26 , 3.34–3.37 , 7.03 , 7.36 , 7.46 , 7.53 , 7.87 , 7.95 , 11.98 . 13C-NMR : δ = 34.34, 45.57, 47.78, 106.55, 115.95, 125.57, 126.04, 126.08, 126.50, 127.99, 138.82, 142.50, 149.30, 151.48, 169.07. ESI-MS (m/z): [M + H]+: 476.05.Yield: 81%, Rf = 0.71, M.P. = 204–208 °C, FTIR : 3304 (N-H), 2827 (C-H), 844. 1H-NMR : δ = 2.93 , 3.23–3.26 , 3.34–3.36 , 7.04 , 7.52–7.55 , 7.76 , 7.96 , 8.06 , 12.06 . 13C-NMR : δ = 34.42, 45.58, 47.64, 104.56, 115.58, 120.89, 125.20, 127.98, 128.99, 129.97, 131.96, 142.30, 143.07, 151.85, 177.84. ESI-MS (m/z): [M + H]+: 510.08.Yield: 77%, Rf = 0.78, M.P. = 142–145 °C, FTIR , obtained from the Anadolu University Research Unit for Experimental Animals, Eskişehir, Turkey, were used in the study. The animals were housed in well-ventilated rooms with a 12/12 h dark/light cycle at a temperature of 24 ± 1 °C. The food in the cages was withdrawn 12 h before the experiments to avoid a possible food interference with the absorption of the test compounds. The experimental protocol of this research has been approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eskişehir, Turkey. The mice were divided into 10 groups of seven animals each. The test compounds were dissolved in sunflower oil and administered (p.o.) to animals at doses of 50 mg/kg in a volume of 0.1 mL . SunflowMeasurements were taken 60 min after the administration of the test compounds or sunflower oil, and 30 min after the administration of morphine .A tail-clip test was used to assess the response of animals to mechanically induced noxious stimuli. A metal artery clamp that applies standardized pressure was placed 2–2.5 cm from the base of the tail and latency for turning and biting the clamp was recorded by a stopwatch . A sensiA hot-plate test was used to assess the reaction of animals against thermal noxious stimuli. The hot-plate device consists of a heated surface kept at a constant temperature of 55 ± 1.0 °C . The animals were placed on the surface of the aluminum plate and pain thresholds were determined before and after the test compounds’ administrations. Paw licking or jumping latencies of each animal were recorded in seconds. In sensitivity tests, animals that failed to show a nociceptive response within 15 s were discarded from the experiments. The cut-off time for this test was accepted as 30 s to avoid an injury to the paws ,55.In both the tail-clip and hot-plate tests, analgesic efficacies of the test compounds were expressed as a percentage of the maximum possible effect (MPE %) using the following equation: v/v acetic acid in a volume of 0.1 mL/10 g. The mice were then placed in transparent boxes and the number of writhing behaviors was recorded for 10 minutes, 5 minutes after acetic acid injections [The acetic acid-induced writhing test was used to evaluate the responses of animals to chemically induced noxious stimuli elicited by i.p. injection of 0.6% jections ,56. ReduThe possible involvement of opioid receptors in the antinociceptive effects of test compounds was examined by mechanistic studies using a non-selective opioid receptor antagonist, naloxone. For antagonism studies, mice were pre-treated with naloxone at a dose of 5.48 mg/kg 15 min before the administrations of test compounds ,41. ThenThe Rota-Rod test device  was used to evaluate the possible effects of test compounds on the motor coordination of animals. Before experiments, mice were trained on the rotating rod of the apparatus set at 16 rpm for 3 consecutive days. Animals that could stay on the rotating mill for more than 180 s were used for the tests. The falling time of the mice was considered as a parameter for motor coordination. The cut-off time for this test was accepted as 10 min ,41,57.p < 0.05.Statistical evaluation of the experimental data was carried out using GraphPad Prism ver. 8.4.3 software . The data used in the statistical analyses were acquired from seven animals in each group. The differences between experimental groups were determined by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey’s test. The results were presented as mean ± standard error of the mean (SEM) and considered statistically significant when GraphPad Prism ver. 8.4.3 software were used for creating the figures.www.pdb.org, accessed date 28 April 2021). Active conformations of these receptors were used. Molecular docking studies were performed using an in silico procedure to define the binding modes of obtained compounds in active regions of opioid receptors. X-ray crystal structures of µ-opioid receptor (PDB ID: 5C1M) , δ-opioiThe structures of proteins were built using the Schrödinger Maestro  interfacTo the best of our knowledge, this is the first study showing that molecules designed to carry thiazole and piperazine moieties together on their structures have convenient pharmacokinetic profiles and show notable antinociceptive efficacies mediated by the opioid receptors at the spinal, supraspinal, and peripheral sites.


Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fatal interstitial lung disease characterized by abnormal transition and proliferation of fibroblasts. The uncontrolled transition of fibroblasts, commonly known as myofibroblasts, are the principal source of the enormous extracellular matrix (ECM) depositing in lung parenchyma, leading to gradual failure of gas exchange and mortality of the patients. However, up to now, rare effective therapeutic strategies have been developed to blockade fibroblast-to-myofibroblast transition (FMT) in IPF.Method: We illustrated that the lungs originated from IPF patients and mice with pulmonary fibrosis are characterized by the overexpression of sushi-repeat-containing protein, X-linked 2 (SRPX2). Further functionality studies identified the pivotal role of SRPX2 in FMT. Mechanistically, SRPX2 was involved in a TGFβR1/SMAD3/SRPX2/AP1/SMAD7 positive feedback loop. Specifically, SRPX2 was upregulated by TGF-β1 in a TGFβR1/SMAD3-dependent manner, after which SRPX2 in turn repressed the expression of AP1, subsequently minimized SMAD7 expression, through which it reduced the formation of inhibitory complex with TGFβR1 and enhanced SMAD signaling pathway to promote FMT and exacerbate pulmonary fibrosis. Notably, intratracheal administration of siRNA-loaded liposomes could effectively suppress the expression of Srpx2 in the lung and remarkably protect mice against BLM-induced pulmonary fibrosis, concomitant with a significant reduction of FMT.Results: Accordingly, these data indicate that Srpx2 plays an essential role in the pathogenesis of pulmonary fibrosis and suggests the strategy aiming at silencing Srpx2 could be a promising therapeutic approach against pulmonary fibrosis in clinical settings. Idiopathic pulmonary fibrosis (IPF), a fatal lung interstitial disease, is characterized by progressive scarring of the pulmonary parenchyma with a median survival of only 2-3 years after diagnosis Current pathogenic theories demonstrate that fibroblast-to-myofibroblast transition (FMT) plays a central role in the pathogenesis of IPF, featured by uncontrolled production of extracellular matrix (ECM) depositing in lung parenchyma To identify more promising targets for the blockade of FMT induced by TGF-β1, herein, we employed deep RNA-Sequence to delineate the transcriptome changes in myofibroblasts. Notably, sushi repeat-containing protein X-linked 2 (SRPX2), a chondroitin sulfate proteoglycan, was strikingly overexpressed in TGF-β1-induced fibroblasts. Although recent studies have revealed the critical role of SRPX2 in the occurrence and progression of various cancers Srpx2 siRNA-loaded liposomes to suppress FMT for the treatment of pulmonary fibrosis. Indeed, both of in vitro and in vivo studies provided compelling evidence that suppression of Srpx2 by siRNA-loaded liposomes significantly protected mice against BLM-induced lung injury and fibrosis, coupled with the pronounced reduction of fibroblast transition in the lung. In a mechanistic study, for the first time, we demonstrated that silencing SRPX2 expression could repress fibroblast differentiation by blocking the AP1/SMAD7/SMAD2/3 signaling axis. Collectively, our data support the notion that Srpx2 plays a critical role in the progression of pulmonary fibrosis and the liposomes-based strategy aiming at silencing Srpx2 could be a viable therapeutic approach against pulmonary fibrosis in clinical settings.Previous studies Antibodies against Collagen type I, α-SMA, SMAD2, SMAD3 were purchased from Cell Signaling Technology . Antibodies against p-SMAD2, p-SMAD3 and p-SMAD2/3 were obtained from Boster Biological Technology Co., ltd . Antibodies against SRPX2, β-ACTIN, SMAD7 and FIBRONECTIN were acquired from Proteintech Group, Inc . Cholesterol and DSPC were acquired from Sigma-Aldrich, Inc.  and mPEG2000-DMG (MW2660) was purchased from NOF Co., Ltd. (Kawasaki Japan). The cationic lipidoid C12-200 was generated through ring opening of epoxides by amine substrates with a previously reported method Lung tissues were collected from patients with non-small cell lung cancer  and patients with IPF (n = 6) in Tongji Hospital after receiving informed consent. IPF was diagnosed according to the American Thoracic Society (ATS)/European Respiratory Society (ERS) consensus diagnostic criteria SRPX2  and a nontargeting control siRNA (named scramble siRNA) were purchased from RiboBio . The specific SRPX2-targeted siRNA sequences were as follows: si-SRPX2_001: 5'-CAG ATG AAA GCT ACA ATG A-3', si-SRPX2_002: 5'-CAG ATG AAA GCT ACA ATG A-3', and si-SRPX2_3: 5'-GAG GAA ATC TTC ACA TTC A-3'. For transfection, Lipofectamine 3000  was used according to previously reported Three siRNAs targeting Liposomes were raised as carriers to encapsulate siRNA. The siRNA was dissolved in citrate buffer  and rapidly mixed with a lipid mixture by vortexing. The lipid mixture was composed of C12-200, cholesterol, DSPC and mPEG-DMG dissolved in ethanol at a molar ratio of 50:38.5:10:1.5. The unentrapped siRNA was removed by ultrafiltration centrifugation. The entrapment efficiency was measured by RiboGreen assay. SiRNA-loaded liposomes were used after being diluted with PBS. The characteristics of liposomes  were listed in Srpx2 siRNA-loaded liposomes, scramble siRNA-loaded liposomes or unloaded liposomes  were injected via intratracheal to the corresponding group of the mice. Eventually, all mice were euthanized 21 days after BLM injection for analysis of pulmonary fibrosis.All male C57BL/6 mice (8-10 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The mice were all housed in a specific pathogen-free (SPF) animal facility at Tongji Hospital in a 12:12 h light/dark photocycle with sterile acidified water and irradiated food. All animal experimental procedures were allowed by the Animal Care and Use Committee at Tongji Hospital. The mice were anesthetized with 1% pentobarbital sodium (60 mg/kg) by intraperitoneal injection, and the 2.5 mg/kg BLM diluted in PBS or PBS alone was administered via intratracheal injection as previously reportedThe left lungs were collected and fixed in 4% neutral buffered paraformaldehyde for 24h at room temperature, followed by embedded in paraffin. Subsequently, the lungs were sliced into 5 µm sections and stained with hematoxylin and eosin (H&E), Sirius red and Masson's trichrome as previously reported A hydroxyproline assay kit  was used to measure the hydroxyproline content of the lungs to assess the lung collagen deposition according to previously reported 2 humidified incubator. The cell culture medium was refreshed every 3 days, and the cells were passaged at a 1:2 ratio every 3-4 days.Human lung tissues from IPF patients and control subjects were minced and allowed to adhere to the bottom of the culture flask. Samples were cultured for 24h after attachment to the culture flask bottom, in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum  at 37 °C in a 5% COin vivo imaging system   as previous reported ex vivo fluorescence imaging and biochemical detection, respectively.DiR-labelled liposomes were constructed as the above method. And then, the prepared liposomes were intratracheal injected to the C57/B6 mice. The mice were then anesthetized and imaged at different time points  by an RNAs from mouse lung fibroblasts after TGF-β1 stimulated for 24 h were extracted using an RNA isolation kit . RNA quality and integrity were determined using the Nanodrop 2000 Spectrophotometer  and Agilent Bioanalyser . RNA-seq libraries were multiplexed and loaded per lane into the Illumina HiSeq flow cell v3. All sequencing protocols were carried out as the manufacturer's instructions using the Illumina HiSeq 1000 and HiSeq control software.SRPX2 forward, 5′- TGG CTG GTT GAT TTT GTA GAG AAA-3′ and reverse, 5′- TAG AAA AGA GTT AGG TGT CAC ATT GAA TAA-3′; human FIBRONECTIN forward, 5′- GAT GTC CGA ACA GCT ATT TAC CA-3′ and reverse, 5′- CGA CCA CAT AGG AAG TCC CAG-3′; human COL1A1 forward, 5′- GAG GGC CAA GAC GAA GAC ATC-3′ and reverse, 5′- CAG ATC ACG TCA TCG CAC AAC-3′; human ACTA2 forward, 5′-GAC GCT GAA GTA TCC GAT AGA ACA CG-3′ and reverse, 5′- CAC CAT CTC CAG AGT CCA GCA CAA T-3′; human ACTB forward, 5′-AGC GAG CAT CCC CCA AAG TT-3′ and reverse, 5′-GGG CAC GAA GGC TCA TCA TT-3′; human AP-1 forward, 5′- CAA ACC TCA GCA ACT TCA ACC-3′ and reverse, 5′- CTG GGA CTC CAT GTC GAT G-3′; human SMAD7 forward, 5′- ATG TTC AGG ACC AAA CGA TCT-3′ and reverse, 5′- GGA TGG TGG TGA CCT TTG G-3′.mouse Srpx2 forward, 5′-ATG AAG TGT TCC AGC GAT GGT GA-3′ and reverse, 5′-TGG CAT ACT CGG GCA GGA CTA C-3′; mouse Fibronectin forward, 5′- GAT GTC CGA ACA GCT ATT TAC CA-3′ and reverse, 5′-CCT TGC GAC TTC AGC CAC T-3′; mouse Col1a1 forward, 5′-TAA GGG TCC CCA ATG GTG AGA-3′ and reverse, 5′-GGG TCC CTC GAC TCC TAC AT-3′; mouse Acta2 forward, 5′-GGA CGT ACA ACT GGT ATT GTG C-3′ and reverse, 5′- TCG GCA GTA GTC ACG AAG GA-3′; mouse Actb forward, 5′-GCC ACA GCA CTC CAT CGA C-3′ and reverse, 5′-GTC TCC GAT CTG GAA AAC GC-3′, mouse AP-1 forward, 5′-AAG ATG GAA ACG ACC TTC TAC G-3′ and reverse, 5′-CTT AGG GTT ACT GTA GCC GTA G-3′; mouse Smad7 forward, 5′-CTG TGT TGC TGT GAA TCT TAC G-3′ and reverse, 5′- GAG ACT CTA GTT CAC AGA GTC G-3′.Total RNA was extracted from fibroblasts and the mice lungs with TRlzol reagent  as previously reported Fibroblasts and lung tissues were homogenized in RIPA lysis buffer. Proteins were subjected to Western blot with the indicated primary antibodies using established techniques For the immunofluorescence experiment, the slides were treated with antibody at 4 °C overnight. The primary antibodies used for staining were as follows: mouse anti-SRPX2 (1:100), rabbit anti-FIBRONECTIN (1:100), rabbit anti-COLLAGEN I (1:100), rabbit anti-α-SMA (1:100), and rabbit anti-p-SMAD2/3 (1:100). All slides were incubated with an Alexa 488-conjugated anti-mouse and Alexa 594-labeled anti-rabbit antibody  after washed with PBS. And then, the nuclei were counterstained with DAPI (4'-6-diamidino-2-phenylindole) for 5 min. Finally, the slides were analyzed under a fluorescence microscope .3 cells/well. Cell proliferation was then measured using the EdU proliferation assay  as previously reported Human pulmonary fibroblasts (HPFs) were purchased from ScienCell Research Laboratories, Inc  and cultured in 96-well plates at a density of 2×104 cells/well. The lower chambers were filled with culture medium containing 10% FBS to function as a chemoattractant. After incubation at 37 °C for 24 h, the cells migrated through the membrane filter and were stained with 0.1% crystal violet .Cell migration assays were performed using Transwell inserts with a membrane with a pore size of 8.0 mm  according to previously reported methods p<0.05 was used to indicate statistical significance.Experimental data are expressed as the Mean ± SEM. All statistical analyses were performed using GraphPad Prism . Two experimental groups were compared using the Student's t test or the Student's t test with Welch's correction for unpaired data. More than two groups were compared using one-way ANOVA with Bonferroni's correction. in vitro results, SRPX2 was indeed up-regulated in IPF patients' lung compared to control subjects  myofibroblasts  were quantified by RT-PCR. Interestingly, the expression level of FMT related genes illustrated a positive correlation with the expression of SRPX2  on day 14 and day 18 (Figure Srpx2 siRNA-loaded liposomes group, as evidenced by the H&E, Masson's trichrome and Sirius red staining (Figure Srpx2 siRNA-loaded liposomes group (Figure  Srpx2 siRNA-loaded liposomes treated mice than that in scrambled siRNA group (Figure The therapeutic effects of Srpx2 siRNA-loaded liposomes displayed attenuated expressions of fibronectin, collagen I and α-SMA (Figure in vitro studies (Figure in vivo studies also indicated that suppression of Srpx2 in lung could effectively repress the phosphorylation of Smad2/3, coupled with a dramatical overexpression of Smad7 and AP-1 (Figure Srpx2 siRNA-loaded liposomes could be a potent therapeutic approach against pulmonary fibrosis via blocking attenuated FMT.To further evaluate the therapeutic effect of the liposomes, we next conducted Western blot, RT-PCR and immunostaining to examine the fibrotic markers in the lung. Consistent to above results, mice administered with A Figure . In lines Figure , the in Srpx2 siRNA reversed BLM-induced lung injury and fibrosis (Figure IPF is a chronic and progressive interstitial lung disease of unknown origin with an average life expectancy of 2-3 years after diagnosis s Figure . Collectvia different signaling pathways Previous studies have demonstrated that SRPX2 could efficaciously regulate proliferation, migration, differentiation and apoptosis of tumor cells Myofibroblasts are critical in the process of pulmonary fibrosis by their secretion of ECM proteins, leading to tissue stiffness and respiratory failure Another critical issue is the underlying mechanisms by which SRPX2 regulated the transition, proliferation and migration of fibroblasts. It is compelling noted that TGF-β1 is one of the most potent and well-studied inducers for FMT via binding with activated TGFβR1 via AP-1 depend manner, as evidenced by inhibiting AP-1 reversed the overexpression of SMAD7 induced by SRPX2 siRNA and activated TGF-β/SMADs signaling pathway. Additionally, similar data were also obtained in the following in vivo studies. Collectively, SRPX2 was upregulated by TGF-β1 in a TGFβR1/SMAD3-dependent manner. After that, the elevated SRPX2 would inhibit the expression of AP1, and subsequently down-regulated the expression of SMAD7, leading to an enhanced TGF-β/SMADs signaling pathway. This positive feedback loop centered on SRPX2 would further promote fibroblasts differentiating into myofibroblasts, exacerbating pulmonary fibrosis.To address the notion that SRPX2 regulated the TGF-β/SMADs signaling pathway, we next examined the expression of SMAD7, which interferes with the phosphorylation of SMAD2 and SMAD3 Srpx2 siRNA could be efficiently phagocytized by the fibroblasts in fibrotic lesions of the lung following intratracheal injection and suppress the expression of Srpx2 for at least 6 days. More importantly, Srpx2 siRNA-loaded liposomes was intratracheally injected into mice during the “fibrotic” phase of the model, which is more applicable and reflective to the clinical management of IPF patients. Since our previous work Srpx2 siRNA-loaded liposomes on macrophages. No obvious difference was detected on the polarization of alternative activated macrophages, which were one of major sources of TGF-β Srpx2 siRNA-loaded liposomes on pulmonary fibrosis is duo to suppression of fibroblasts behavior, not macrophages activation. Overall, intratracheal injection of Srpx2 siRNA loaded liposomes could block TGF-β signaling pathway and subsequently decelerate the process of FMT, contributing to the protection of mice from BLM-induced lung injury and fibrosis, which provide an available, safe and effective therapeutic approach for IPF.Although numerous clinical trials have been carried out to characterize viable drugs for IPF, unfortunately, no effective therapeutic approach was currently available to cure or even halt the progression of IPF Srpx2 siRNA significantly reduced the expression of Srpx2 and then suppressed FMT, contributing to the improvement of the pulmonary fibrosis induced by BLM. Briefly, our data supported that intratracheal administration of Srpx2 siRNA loaded liposomes could be a promising therapeutic approach against pulmonary fibrosis in clinical settings.In conclusion, we firstly demonstrated that SRPX2 was overexpressed in the fibroblasts originated from IFP patients and pulmonary fibrosis mice. Mechanistically, SRPX2 was upregulated by TGF-β1 canonical signaling pathway during the process of FMT. In addition, the elevated SRPX2 would inhibit the expression of AP1, and then attenuated SMAD7 expression, forming a positive feedback loop to enhance TGF-β/SMADs signaling, which could finally promote FMT and exacerbate pulmonary fibrosis. Inspiringly, administration of liposomes carrying Supplementary figures.Click here for additional data file.


Moreover, the PQS ELISA here reported has been foundto be robust and reliable, providing accurate results in culture media.The technique allowed us to follow up the PQS profile of the releaseof bacterial clinical isolates obtained from patients of differentdisease status. A clear correlation was found between the PQS immunoreactivityequivalents and the chronic or acute infection conditions, which supportsthe reported differences on virulence and behavior of these bacterialstrains due to their adaptation capability to the host environment.The results obtained point to the potential of the PQS as a biomarkerof infection and to the value of the antibodies and the technologydeveloped for improving diagnosis and management of P. aeruginosa infections based on the precise identificationof the pathogen, appropriate stratification of the patients accordingto their disease status, and knowledge of the disease progression.Quorumsensing (QS) is a bacterial cell density-based communicationsystem using low molecular weight signals called autoinducers (AIs).Identification and quantification of these molecules could providevaluable information related to the stage of colonization or infectionas well as the stage of the disease. With this scenario, we reporthere for the first time the development of antibodies against thePQS , the main signaling molecule fromthe  Accordingto the U.S. Centers for Disease Control and Prevention (CDC), about51,000 P. aeruginosa HAI cases occureach year in U.S. hospitals. This pathogen has developed a vast adaptiveresponse and significant resistance to innate immune effectors andto antibiotics, particularly during chronic infections. Such is thecase in patients suffering from cystic fibrosis (CF), a disease causedby a genetic disorder that predominantly affects the mucociliary clearanceof the respiratory tract,2 creating anideal scenario for opportunistic pathogens such as P. aeruginosa. This pathogen colonizes the lungsof these patients at the age of 2–3 years old and ends up persistingin 60–80% of CF patients as a chronic infection, compromisingtheir life and frequently ending in respiratory failure and prematuredeath.3 On the other hand, antibiotic therapyrarely leads to elimination of a pathogen from chronically infectedCF respiratory airways. In 2017, multidrug-resistant P. aeruginosa caused an estimated 32,600 infectionsamong hospitalized patients and 2700 estimated deaths in the UnitedStates.4Healthcare-associatedand community-acquiredinfections  have become a major burdenfor public health worldwide,P. aeruginosa.5 The lackof reliable tools for rapid diagnostic of bacterial infections hascontributed to the overprescription and misuse of broad spectrum antibiotics,strengthening the generation of multidrug-resistant bacteria.6 Current diagnostic methods based on a cultureplate require several days to obtain conclusive results, which oftenis an unaffordable time for effectively treating the infection.7 Many diagnostic alternatives have emerged aspotential complements of the standard techniques, providing more completeand accurate results. For instance, matrix-assisted laser desorptionionization–time of flight–mass spectrometry (MALDI-TOF-MS)analysis has been successfully used to characterize the protein footprintfrom bacterial isolates of CF.8 Also, polymerasechain reaction (PCR) methods have appeared as interesting alternativesto identify pathogens.9 However, thesetechniques require complex selection of the targets, tedious and time-consumingextractions, highly trained personnel, and/or expensive equipment,10 which prevent their wide implementation on clinicalsettings. Point of care (PoC) devices may be a promising option infuture routine clinical diagnostics, providing rapid reliable responsesand being low cost and user-friendly to use. PoC performance wouldallow us to change the management of the patient and would providefaster diagnosis than conventional methods, ideally in almost everyplace and situation. Most of the time, these devices rely on the useof a biorecognition element that specifically binds the correspondingbiomarker target,11 such is the case ofantibodies.Recently, the World HealthOrganization (WHO) has published a listdeclaring the urgent need of finding new antibiotics for the treatmentof infections caused by resistant bacteria, such as carbapenem-resistant 13 Because of their key role in the development of pathogenesis, thesignaling molecules of the quorum sensing (QS) system could be a potentialbiomarker of infection. QS is a bacterial communication mechanismbased on the release of low molecular weight signals that regulatethe coordinated genetic expression of a wide array of physiologicalactivities.14 These signals, also calledautoinducers (AIs), promote their own biosynthesis and their concentrationincrease as a function of population density. At a particular AI concentrationthreshold, the genetic expression of decisive factors such as thesecretion of virulence factors and biofilm formation is triggered. P. aeruginosa QS is a well-studied communicationnetwork composed of four systems  performing in acomplex hierarchical manner. Particularly, the quinolone-based pqs system, responsible for the regulation of virulencefactors including pyocyanin, elastase, lectin, and rhamnolipids,15 has particularly attracted the attention ofthe scientific community due to its QS-dependent and independent functions.16 Its main signaling molecule, 2-heptyl-3-hydroxy-4(1H)-quinolone or the PQS ,specific for P. aeruginosa, is responsiblefor important functions besides its signaling activity. For instance,the PQS mediates cytotoxicity and has been indicated to balance thegrowth and death in P. aeruginosa populations.17 The PQS also mediates iron acquisition due toits strong chelating character and its activity to prompt the expressionof genes related to the biosynthesis of the siderophores pyoverdineand pyochelin.19 Furthermore, the PQS promotesthe formation of outer membrane vesicles (OMVs) due to its remarkedlipophilic character. The molecule induces the curvature of the bacterialmembrane and resides as an integral component, maintaining its biologicalactivity and achieving the communication between bacteria throughthe aqueous media.20 Eventually, the PQSwas also found to modulate the host immune response by dysregulatingseveral defense mechanisms and cytokine expression.21Host–pathogen interaction involves the releaseof a largeamount of molecules, which have potential as biomarkers for diagnosis,patient stratification, disease monitoring, or for guiding antibiotictreatment as being the subject of investigation.23 such as, for example, in sputum,approximately at the micrometerrange by thin-layer chromatography (TLC) analysis,24 although other more selective techniques found it at concentrationsin the low nanometer range.25 While massspectrometry has been the most commonly used technology to quantifythese molecules,27 electrochemical28 or bioreporter methods29 havealso been reported. These measurements require complex sample treatmentmethods such as extraction using organic solvents and tedious purificationor enrichment steps to accomplish the necessary detectability. Onthe other hand, only in very few cases, these alkylquinolones havebeen measured in clinical samples, partially due to the increasedsample complexity and low detectability attained by some of thesetechniques. With this scenario, we have focus our attention on thedevelopment of specific antibodies against the main signaling moleculeof the pqs QS system, with the final aim of developingimmunochemical diagnostic technologies suitable to assess the potentialvalue of these specific QS molecules as biomarkers of infection. Inthis paper, we report, for the first time, the development of a highlysensitive microtiter-based enzyme-linked immunosorbent assay (ELISA)to quantify the PQS and its use to analyze the concentration levelsof this molecule in culture media where clinical isolates obtainedfrom infected patients have been grown. Preliminary data point tothe potential value of this QS molecule as the biomarker to diagnose P. aeruginosa infection and to stratify patientsin respect to the stage of infection.The PQS has been found in several body fluids ofinfected patients,Supporting Information.See the (13) was synthesized following a similar approach to thatdescribed by Reen et al.30 for the synthesisof the PQS through a five-step synthetic pathway from 3-(4-aminophenyl)propanoicacid and methyl 3-oxodecanoate (3), obtained by a nucleophilicreaction of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid)with octanoyl chloride and subsequent methanolysis. All the intermediatesand the final product were purified and characterized by nuclear magneticresonance (NMR) spectroscopy and ultraperformance liquid chromatography(UPLC)-MS/MS.31The hapten3-propanoic acid (13) in anhydrous DMF (400 μL) was cooled to 4 °C. Afterward,tri-n-butylamine  andisobutyl chloroformiate  were addedto the hapten solution and the mixture was left stirring for 15 minat 4 °C and 30 min at room temperature. Then, 200 μL ofthe reaction mixture was slowly added over the protein solution  or keyhole limpet hemocyanin (KLH), 2.5 mg/mL,2 mL in 10 mM phosphate buffered saline (PBS)). Then, the mixturewas left stirring for 2 h at RT and overnight at 4 °C withoutagitation. The bioconjugates were purified by dialysis against 0.5mM PBS (5 × 5 L) and Milli-Q water (1 × 5 L) and storedfreeze-dried at −80 °C. A small fraction (20 μL)of PQS-BSA was kept for MALDI-TOF-MS analysis, rendering a haptendensity of 17 haptens per molecule of BSA (see Table S1). Hapten densities of PQS-BSA and PQS-KLH were alsoassessed by fluorescence, measuring the intensity of the emissionat 445 nm, of the native protein and the bioconjugates, and interpolatingthe value on a calibration curve build with the PQS hapten, renderingthe same value of previous MALDI-TOF-MS analysis .A solution of the PQS hapten Supporting Information.See the 2SO4 solution(50 μL/well), and the absorbance was read at 450 nm.Microtiter plates were coated withthe HHQ-BSA conjugate in coating buffer overnight at 4 °C and covered with an adhesive plate sealer.The day after, plates were washed with PBST (4 × 300 μL/well)and solutions of PQS standards  followed by the As385  addition and the microplates were left without agitationfor 30 min at room temperature. After another washing step, a solutionof diluted goat anti-rabbit IgG-HRP (1/6000 in PBST) was added (100μL/well) and incubated for 30 min at room temperature. The plateswere washed again, and the substrate solution (100 μL) was addedand left for 30 min at room temperature in the dark. The enzymaticreaction was stopped by adding 4 N HPerformance of the assays wasevaluated through the modification of different physicochemical parameters, solubilitywith addition of organic solvents, competence time, incubation time,or cation complexation by EDTA) in the competitive step.50 value was used to calculate the cross-reactivityaccording to the following equation CR (%) = IC50(crossreactant)/IC50 × 100.HHQ, PQS, and HQNO calibrationcurves (10 μM to 1 pM) were built in PBST and measured on theAs385/HHQ-BSA ELISA following the procedure described above. The standardcurves obtained were fitted to the four-parameter equation mentionedabove, and the ICP. aeruginosa isolates were selected and stored frozen at −20 °C.Prior to the analyses, P. aeruginosa isolates were cultured overnight at 37 °C in blood agar plates.The day after, a portion of the grown bacteria was diluted in Mueller–Hinton(MH) culture broth (3 mL) and shaken at 37 °C. When the opticaldensity at 600 nm (OD600) reached a value of 0.2–0.3,a dilution (1/1000 in 20 mL of MH) was performed and the resultingsolution was shaken at 37 °C. Aliquots were taken at the selectedtimes for measurement of OD600, for colony forming unit(CFU) calculation, and measurement of the PQS by the ELISA. The restof the isolates were grown using the same experimental procedure,yet the aliquots were extracted just at a selected time of 8 h. PQSconcentrations measured by the ELISA were expressed as PQS immunoreactivityequivalents (IRequiv.) due to the potential specific interferencescaused by other alkylquinolones of different chain lengths potentiallypresent in the culture media.Clinicalisolates were obtained from lowerrespiratory tract samples, mainly sputum specimens, of patients diagnosedof acute or chronic respiratory infections. Clinical samples werecultured in MacConkey agar, blood agar, and chocolate agar and incubatedfor up to 5 days at 37 °C. Culturebroth samples were analyzed following the same procedure as describedabove except for coating the plates with HHQ-BSA at 0.04 μg/mL and diluting As385 64,000 timeswith PBST-EDTA. The standards used to build the standard curves wereprepared in MH broth diluted 10 times with PBST-EDTA, and the sampleswere diluted 10 times with PBST-EDTA before measuring them with theELISA. To assess the accuracy of the method, blind spiked samplesusing diluted MH culture broth (1/10) were prepared and measured usingthe above-described ELISA for three different days and three replicateswithin the same day. The results are expressed as the mean of allreplicates.Understandingquorum sensing (QS) is key to apprehend about pathogenesis.QS depends on specific signaling molecules or autoinducers, whichare responsible for triggering the expression of a series of genes,biosynthesizing virulence factors, and favoring biofilm progression.Rapid detection of these signaling molecules could give cliniciansan early indication of infection or knowledge about the stage of thedisease.Over the last few years, biosensors and PoC deviceshave emergedas promising alternatives for a more rapid and efficient detectionof biomarkers of the disease. Among them, antibody-based technologieshave demonstrated their capability of providing accurate and reliablediagnostic results thanks to their great affinity, which allows usto develop highly sensitive technologies and their specificity. However,accomplishing high quality antibodies with the necessary affinityand specificity is a crucial factor, which is mainly dependent onthe immunogen and of the strategy used to produce such antibodies.P. aeruginosa to regulate virulence .34The pseudomonasquinolone signal (PQS) is an autoinducer used specifically by ence see 1 and for35 basedon two sequential modifications of the C-3 position in the PQS biologicalprecursor, 2-heptyl-4-quinolone or HHQ. Hapten synthesis (see (9). The aniline derivative was subsequentlyreacted by amination reaction with β-ketoester (3) in the presence of a catalytic amount of p-toluenesulfonic acid to obtain the intermediate (10). Afterward,the quinolone derivative (11) was prepared through Conrad–Umpachthermic cyclization in diphenyl ether at 270 °C. Formylationof the C-3 position through the Duff reaction36 allowed us to obtain the quinolone derivative (12) asthe precursor of the desired hapten. Concomitant hydrolysis of themethyl ester group occurred; however, this fact no longer affectedthe synthetic procedure. Finally, oxidation of the C-3 position throughDakin reaction allowed us to obtain the PQS hapten (13). This reaction relies on a characteristic hydrogen peroxide nucleophilicaddition that promotes the -aryl migration and elimination, subsequentlyobtaining an hydroxyl group directly bonded to the previously formylatedposition.37 Overall, the pure proposedPQS proposed hapten (13) was obtained in five syntheticsteps with an overall yield of 16%.The synthesis of the PQS hapten was inspired from a strategy previouslydescribed by Pesci and co-workersesis see 1 started38 Hapten bioconjugateswere obtained with a high density ratio , as demonstrated by MALDI-TOF-MS and fluorescenceanalysis .The PQS hapten was usedto prepare KLH and BSA protein bioconjugatesfollowing the anhydride mixed method. This method relies on the carboxylicacid activation using a chloroformate and a sterically hindered base,forming an anhydride that afterward reacts with the lysine residuespresent in the protein.Table S2), from which the combination of As385/HHQ-BSA was selected for furtherstudies due to the higher detectability in further experiments.Antisera  were increased against PQS-KLH in female New Zealand whiterabbits. The avidity of obtained As for the BSA conjugates (HHQ-BSAand PQS-BSA) and the competition of the PQS for the binding to theantibodies were assessed by combined experiments, comparing the absorbanceof the assay in the presence and absence of the analyte. After a preliminaryselection, the most promising combinations were analyzed by two-dimensionaltitration experiments. The first set of experiments allowed us toselect the best As/bioconjugate combinations for competitive assaysbased on the largest reduction of the signal when the PQS was presentin different combinations, while the second one allowed us to selectthe concentrations of the immunoreagents providing the best ratio/noisesignal. As a result, four different As/bioconjugate combinations wereevaluated in a competitive format . A significant variationof the assay features by introducing a preincubation step was notobserved, and the optimum competition time was found to be 30 min.The performance of the assay under different physicochemical conditionssuch as the effect of time, pH, concentration of a non-ionic surfactant,ionic strength, or the presence of an organic solvent was evaluated, while the IC50 value wassignificantly lower (better detectability) than under the standardconditions using a buffer with 0.05% of this detergent. However, sinceit is known that the presence of a small amount of Tween 20 helpsin minimizing nonspecific interactions and solubilizing the analyte,it was decided to keep a concentration of just 0.01%. The detectabilitywas slightly better at conductivity values greater than 15 mS/cm;however, the Amax decreased substantially,and for this reason, it was decided to keep the conductivity at 15mS/cm for further experiments. Furthermore, it was added to the assaybuffer (0.1 mM) of EDTA as a result of the accuracy experiments (explainedin the section below).The assay was able to workat pH values between 4.5 and 9.5, althougha slightly better performance was observed at pH 7.5 based on itsdetectability and maximum absorbance. The addition of an organic solventsuch as DMSO did not produce a significant enhancement; although the P. aeruginosa bacterial cultures (μM) and in the same range of concentrations(low nanometer range) compared to the ones found in sputum samplesby Abdalla and co-workers.39 Furthermore, the detectability achieved with the As385/HHQ-BSA ELISAis greater than those obtained with other techniques such as thosebased on bioreporters40 or electrochemical methods.28 Regarding LC-MS/MS methods, the ELISA reportedhere allows us to obtain higher or similar detectability without includingadditional preconcentration steps. These methods require the use ofsolid phase extraction and/or organic solvent extraction steps, endingup in a 20 times concentration factor of the sample for obtainingthe reported LOQs.39Under these conditions,the assay showed an LOD of 0.17 ± 0.01 nM and a dynamic rangecompressed between 0.53 ± 0.04 and 24.37 ± 3.18 nM see 2, which P. aeruginosapqs QS system. However, other quinolones produced by this pathogen havealso an important role in communication or interspecies interaction.The other two molecules are HHQ, the precursor of the PQS in the biosyntheticpathway and involved in the signaling process, and HQNO, an N-oxidequinolone able to inhibit the electron transport chain in other bacteriashowing remarkable antistaphylococcal properties.41 The only structural difference between HHQ, HQNO, and thePQS is one oxidized position (C-3 or N-1), the reason why the recognitionpattern by the As385/HHQ-BSA ELISA had to be assessed. Despite thesimilarity between the different molecules, the percentage of cross-reactivitywas less than 2% for HQNO (IC50 = 236.2 nM) and about 13%in the case of HHQ (IC50 = 27.2 nM) (see Table S5). Moreover, additional specificity experiments wereperformed in which calibration curves of the PQS were built underthe presence of constant concentrations of either HHQ or HQNO . It allowed us to evaluate the potentialcross-reactivity for each point of the calibration curve at differentconcentrations of cross reactants. The experiments showed that, indeed,the percentages of cross-reactivity were not constant and diminishedwhen the concentrations of the PQS increased. Hence, the ELISA developedrecognized a much better PQS despite the great structural similaritiesof the other two autoinducer molecules of the pqs system, which pointed to the possibility to quantify specificallythe PQS from samples where all three quinolones will be present. Specificitystudies using other molecules structurally related  and non-structurally related-thiazole-4-carbaldehyde,the main signaling molecule of the iqs QS system),potentially present in clinical or bacterial culture samples, werealso performed. In all cases, the As385/HHQ-BSA ELISA did not respondto concentration values up to 10 μM of all mentioned molecules,and thus, the CR was assumed to be less than 0.01% for all of them.The PQS is the most important and active signalingmolecule inthe Table S3, where the percentage of the coefficient of variation (%CV)at three different concentrations is shown . The reproducibility is only slightlylower when measuring concentrations close to the limit of quantificationduring different days, but even at those very low concentration values,the %CV is below near 10% on intra-plate and inter-plate studies.As385/HHQ-BSA ELISA intra-plate, inter-plate, and inter-day precisionwere assessed, as it can be observed in P.aeruginosa was grown. The total amount of exo-productsand production profile of QS molecules such as the PQS can vary amongstrains coming from different infection types or infection status3 and might have clinical relevance. However, suchtypes of samples may contain a variety of components from the mediaor from the bacteria, which may interfere with the assay. For thisreason, on the first instance, the matrix effect caused by measurementin Mueller–Hinton (MH) culture broth, frequently used to grow P. aeruginosa strains, was evaluated. These mediacontain nitrogenous compounds, vitamins, starch, and amino acids,which added to the products released by the bacteria itself such asvirulence factors, proteins, and other exo-products, which could makethe sample quite complex. To estimate the extension of this effect,calibration curves were built in MH culture media, at different dilutionfactors, and measured with the As385/HHQ-BSA ELISA. The resultingcalibration curves and the effect caused by MH in the assay performancecan be seen in 50 value was 6.05 ± 0.16 nM, while the obtainedLOD was 0.36 ± 0.14 nM  in a non-deliverable manner19 causes an iron starvation response and stimulates a concentration-dependentincrease production of iron scavenging siderophores such as pyoverdineand pyochelin. PQS-Fe3+ has been found to accumulate inthe membrane of the cell, acting as a storage molecule. Consideringsuch behavior, the PQS might be forming complexes with the cationspresent in MH culture broth. To probe this hypothesis, the calibrationcurve was run in the presence of different concentrations of ethylenediaminetetraaceticacid (EDTA), a powerful ion chelator able to displace the divalentcation complexation by the PQS . The addition of EDTA caused a substantial increase of the maximumabsorbance; nevertheless, it produced an increase in detectability.In light of these results, we assessed the effect of EDTA in the buffer(PBST-EDTA) on the assay accuracy. As it is shown in Accuracy of the assay was evaluated by preparing blind spiked samplesin MH culture broth and measuring them with the developed ELISA. Interestingly,the results showed a critical underestimation of the spiked PQS concentrations3. We hyp the PQS S4. The aP. aeruginosa airway infection, respectively, were initially measured.With these results, we addressed the investigation of thePQS productionprofile of clinical isolate growth on MH broth media. Since previouslyperformed experiments pointed at a different production profile ofHHQ, the PQS biosynthetic precursor, depending on whether the clinicalisolates belonged to patients with a chronic or an acute infection,and the PQS profiles of the release of isolates from patients withan acute (PAAI6) and a chronic (PACI6) 600 andthe calculated CFUs. The decrease in the OD600 at 48 hof growth observed in PAAI6 might be explained by the prolonged exposureto high concentrations of self exo-products with lytic activity, suchas pyocyanin and the PQS.44Interestingly,similarly to HHQ, PQS quantifiable levels were reachedafter 5 and 12 h of growth for the clinical isolates PAAI6 and PACI6,respectively, while after 48 h, the PQS IRequiv. value of PAI6 washigher than that of PAAI6 see 4, as alrP. aeruginosa infectionwith distinct severity degrees. For this purpose, clinical isolatesfrom 12 patients with acute or chronic infections were grown in MHat 37 °C. After 8 h (time chosen as a compromise based on theabove-described behavior), sample aliquots were taken and measuredwith the PQS ELISA developed. As it can be observed in The next set of experiments was seeking to demonstrate that thiswas a general behavior for clinical isolates coming from patientssuffering from P. aeruginosa adapts to the host environment by evolving toward a state of reducedbacterial invasiveness that favors bacterial persistence without causingoverwhelming host injury. In that respect, the results found withthe here reported PQS ELISA are in agreement with this situation.The low levels of the PQS released by the isolates from chronic infectedpatients would justify a potential lower production of virulence factors.From a diagnostic perspective, in addition to identifying the microorganismscausing the disease, being able to distinguish between a chronic andan acute infection is very relevant to apply appropriate therapeuticstrategies and improve disease management.In light of this outcome, it seems clear that the concentrationof QS molecules released to the media can be correlated with the typeof infection or the disease status of the patient. It has been oftenreported that the behavior of strains inducing acute or chronic infectionsis substantially different in terms of virulence and exo-product releasecaused by an adaptation to the hostile host environment. Thus, onchronic lung infections, pqs QSsystem of P. aeruginosa have been reported.The produced antibodies were used to develop the indirect competitiveAs385/HHQ-BSA ELISA for the quantification of the PQS, achieving adetectability that is within the range of concentrations found inbiological and clinical matrices. The results obtained when measuringthe PQS IRequiv. released by clinical isolate growth on MH culturebroth demonstrated the ability of the As385/HHQ-BSA ELISA to stratifypatients depending on the state of the disease. If this immunochemicalstrategy is applied for a higher number of QS molecules and/or infectionbiomarkers, then it might provide interesting information for diagnosticor prognostic purposes as well as about the pathogenic strain, thestatus of the patient, and/or the progression of the disease. In sucha case, the technique developed in this work could be envisaged asa future complement of the standard clinical analysis and help clinicianson taking decisions about the treatment and the management of infectedpatients. On the other hand, the obtained limits of detection anddynamic ranges led us to consider applying the method for the directquantification of the PQS in clinical respiratory samples from infectedpatients and its evaluation as the biomarker of infections. Eventually,the versatility and specificity of antibodies would also allow usto implement the developed technique in a wide variety of sensorsand/or PoC devices, supporting the rapid and straightforward diagnosticof infections caused by P. aeruginosa and subsequently improving the management of the patients.Inthis work, for the first time, antibodies against the most relevantand biologically active quinolone from the